Difference between revisions of "Part:BBa K1766011"

 
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The bacterial antigen receptor (BAR) was created in the hope of making a receptor able to signal binding to an antigen. By making a chimera between a histidine kinase (EnvZ) and a affibody molecule we would have a receptor capable of detecting protein biomarkers. The construct would then use the use the EnvZ-OmpR two-component regulatory system to signal biomarker binding and activation of the BAR.
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The osmoregulator EnvZ was chosen a the scaffold for the BAR as it is a well characherized protein that has been used in other many  iGEM projects. One particularly interesting aspect of EnvZ is that functional chimeras between it and other histidine kinases have been made. Specifically protein containing a HAMP domain responsible for signal progression seem to be suitalble for fusion proteins which is refered to as a “control cable”[1]. For example, Cph8 (BBa_K1017301 and BBa_I15010), a Cph1-EnvZ chimera is already available in the registry. Also in a reasent article [2] the activation and inactivation of EnvZ by moving aromatic residues was studied and later employed on a Tar-EnvZ chimera [3].
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One troubling aspect on EnvZ is that the periplasmic domain has not been properly characherized or the structure determined. Structure prediction software recoognized what seems to be a PAS domain that could be involved in the signaling of EnvZ. In this PAS domain we found regions that seemed suitable for inserting a biomarker binding protein.
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For binding the portein biomarker we choosed the small and stable affibody molecule. Affibody molecules consists of three helixes containging changeable residues for creating affinity to a certain protein. This has been used to create affibodies towards manny different proteins such as the HER2 (human epidermal growth factor receptor) protein. This variablilty and the stability of the structure would mean that the affibody could be switched to detect other proteins as well.
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Construct 3 was created by replacing smaller part of the PAS domain with the affibody Z:HER2:342. This resulted in 27residues in EnvZ were exchanged for 58 residues in the affibody. The PAS Beta sheet/Coil I region was kept as this was thought to effect dimerization between constructs.
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The construct has been studied for expression, presence in membrane and affinity towards HER2.
 
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===Usage and Biology===
 
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===Functional Parameters===
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===References===
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[1] Parkinson, J. S. (2010) <i>Signaling mechanisms of HAMP domains in chemoreceptors and sensor kinases</i>. Annu. Rev. Microbiol. 64, 101− 122
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[2] Nørholm M. H. H., von Heijne G., and Draheim R. R. (2014) <i>Forcing the Issue: Aromatic Tuning Facilitates Stimulus-Independent Modulation of a Two-Component Signaling Circuit</i>. ACS Synth. Biol. 2015, 4, 474−481
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[3] Yusuf R. and Draheim R. R. (2015) <i>Employing aromatic tuning to modulate output from two-component signaling circuits</i>. Journal of Biological Engineering, 9:7 
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Revision as of 20:32, 14 September 2015

BAR construct 3

The bacterial antigen receptor (BAR) was created in the hope of making a receptor able to signal binding to an antigen. By making a chimera between a histidine kinase (EnvZ) and a affibody molecule we would have a receptor capable of detecting protein biomarkers. The construct would then use the use the EnvZ-OmpR two-component regulatory system to signal biomarker binding and activation of the BAR.

The osmoregulator EnvZ was chosen a the scaffold for the BAR as it is a well characherized protein that has been used in other many iGEM projects. One particularly interesting aspect of EnvZ is that functional chimeras between it and other histidine kinases have been made. Specifically protein containing a HAMP domain responsible for signal progression seem to be suitalble for fusion proteins which is refered to as a “control cable”[1]. For example, Cph8 (BBa_K1017301 and BBa_I15010), a Cph1-EnvZ chimera is already available in the registry. Also in a reasent article [2] the activation and inactivation of EnvZ by moving aromatic residues was studied and later employed on a Tar-EnvZ chimera [3].

One troubling aspect on EnvZ is that the periplasmic domain has not been properly characherized or the structure determined. Structure prediction software recoognized what seems to be a PAS domain that could be involved in the signaling of EnvZ. In this PAS domain we found regions that seemed suitable for inserting a biomarker binding protein.

For binding the portein biomarker we choosed the small and stable affibody molecule. Affibody molecules consists of three helixes containging changeable residues for creating affinity to a certain protein. This has been used to create affibodies towards manny different proteins such as the HER2 (human epidermal growth factor receptor) protein. This variablilty and the stability of the structure would mean that the affibody could be switched to detect other proteins as well.

Construct 3 was created by replacing smaller part of the PAS domain with the affibody Z:HER2:342. This resulted in 27residues in EnvZ were exchanged for 58 residues in the affibody. The PAS Beta sheet/Coil I region was kept as this was thought to effect dimerization between constructs.

The construct has been studied for expression, presence in membrane and affinity towards HER2. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 402
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 236
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Parkinson, J. S. (2010) Signaling mechanisms of HAMP domains in chemoreceptors and sensor kinases. Annu. Rev. Microbiol. 64, 101− 122
[2] Nørholm M. H. H., von Heijne G., and Draheim R. R. (2014) Forcing the Issue: Aromatic Tuning Facilitates Stimulus-Independent Modulation of a Two-Component Signaling Circuit. ACS Synth. Biol. 2015, 4, 474−481
[3] Yusuf R. and Draheim R. R. (2015) Employing aromatic tuning to modulate output from two-component signaling circuits. Journal of Biological Engineering, 9:7